Several molecular genetic studies have been conducted by our laboratory staff over the past year. A collaboration with epidemiologists at the NCI continues to study the interaction between molecular and classical risk factors of oral cancer. We are using biological samples already collected in a case-control study of oral and pharyngeal cancer conducted in Puerto Rico. The study focused on Puerto Rico because of the especially high incidence of oral cancer known to occur there. Our analyses thus far have focused on applying highly sensitive techniques for detection of human papilloma virus using polymerase chain reaction methods. We extracted DNA from 150 paraffin embedded sections of tumor tissue. Preliminary results indicate that this virus is infrequently present in these oral cancers from Puerto Rico. We have also characterized a polymorphism at the aryl hydrocarbon receptor, a key mediator of the detoxification pathways involving P450 cytochrome. Statistical analyses suggest that this molecular variant may interact with exposure to risk factors such as smoking and drinking in influencing risk of oral cancer. We have been developing plans and protocols for performing molecular analyses of several additional DNA polymorphisms that have been shown to be associated with risk of other forms of cancer. Statistical analyses will be performed for candidate genes using this collection of study subjects. Molecular analyses of p53, HPV and other appropriate markers such as those in regions known to be associated with Loss of Heterozygosity (LOH) will also be conducted on available tumor tissues. We have also completed analyses evaluating the possible effect of mouthwash on oral cancer risk in our Puerto Rico Study population and found little if any evidence for increased risk associated with mouthwash use. A second molecular epidemiological study of oral cancer is being conducted in Athens, Greece, in collaboration with Dr. Anthanasios Zavras, a dentist and Ph.D. student in the Department of Oral Health Policy and Epidemiology at the School of Dental Medicine, Harvard University. We are using a case-control design, based on recruitment in hospitals in Athens. Our laboratory's role is to perform the molecular and statistical analyses for this study, as well as provide guidance about the study's design and performance. Risk factors, family histories of cancer and biological samples (oral rinses and brushings, tumor biopsy or surgical specimens (if available) and blood spots on filter paper) are being delivered to our laboratory for patients and controls. We completed an analysis of alcohol and tobacco risk factors in this population and our manuscripts reporting our findings are now in press. We have evaluated genes potentially responsible for oral cancer, such as those of the cytochrome P450 pathway and in a preliminary analysis found a lack of association with the CYP1A1 gene in this population. In addition to studies of DNA polymorphisms at many other genes involved in detoxification of carcinogens, we plan to also evaluate the role of human papilloma virus, including interaction with variation at HLA genes. Molecular analyses will be conducted to assess mutations in oncogenes and tumor suppresser genes in tumor tissues, and these results will be correlated with the subjects' exposure to risk factors such as smoking and drinking as well as protective factors including the consumption of fresh fruits and vegetables. Statistical analyses will be performed for candidate genes using this collection of study subjects. Molecular analyses of p53, HPV and other appropriate markers such as those in regions known to be associated with Loss of Heterozygosity (LOH) is being conducted on available tumor tissues. In Taiwan, we are collecting multiplex families, and also over 100 simplex families with both parents and at least one unaffected sibling of the OC case available for sampling. Simplex families such as these usually involve OC cases with relatively early age of disease onset (since parents are still alive) and might, therefore, be enriched for high genetic susceptibility as has been the case for early onset forms of other cancers. In addition, such sampling designs are suitable for family-based transmission disequilibrium gene mapping approaches which may provide some advantages over traditional case-control designs. As described above for NPC, we are recruiting from 68 OC families in Taiwan using both the Cancer Registry (4,238 letters sent, responses received from 762 or 18%) and hospital clinics. In the course of this family ascertainment effort, we have also distributed 753 segregation analysis questionnaires for OC, and have received 429 (57%) completed. Analyses of these data will compliment our gene mapping studies. Biospecimens have been collected from over 600 subjects from Taiwan, and risk factor and family history questionnaires are being coded for key entry. Sections from paraffin-embedded tumor tissue are being obtained from approximately half of these OC cases for molecular analyses. A new collaboration with collaborators at the International Agency for Research on Cancer (IARC) in Lyon France has provided our laboratory with access to over 3,500 biospecimens and risk factor data from the ongoing IARC Multinational Oral Cancer Study. This resource will provide unprecedented power for genetic epidemiology research for this complex disease. Our laboratory has been carrying out DNA extraction from buffy coat samples, and analyses of SNPs in cancer susceptibility and nicotine/alcohol addiction candidate genes, and statistical analyses in partnership with IARC investigators. Analyses of a SNP in the ADH3 gene had suggested an effect on OC risk in our Greek sample that is in the opposite direction of that reported previously in the Puerto Rico OC Study. This example illustrates how unstable findings of SNP associations have so often been, with different studies either failing to replicate or even suggesting associations in opposite directions. We believe it will be especially important that adequate numbers of subjects are employed in future studies, to minimize the pursuit of too many false positive findings that turn out to be artifacts of small samples. This is why we have devoted so much effort to building adequate research resources through long term, international collaborative efforts.